RESUMEN
OBJECTIVE: To evaluate the potential of ADAMTS-4 (aggrecanase -1) activity in synovial fluid (SF) as a biomarker of knee injury and joint disease. DESIGN: We have measured ADAMTS-4 activity in the synovial fluid of 170 orthopaedic patients with different degrees of joint pathology, using a commercial ADAMTS-4 fluorescence resonance energy transfer (FRET) substrate assay. Patients were classified at arthroscopy as (i) macroscopically normal, (ii) with an injury of the meniscus, anterior cruciate ligament or chondral/osteochondral defects or (iii) with osteoarthritis, and the influence of independent factors (age, patient group, effusion and synovial inflammation) on ADAMTS-4 activity levels was assessed. RESULTS: In most patients (106/170) ADAMTS-4 activity was undetectable; ADAMTS-4 ranged from 0 to 2.8 ng/mL in synovial fluid from patients with an injury, 0-4.1 ng/mL in osteoarthritic patients and 4.0-12.3 ng/mL in patients with large effusions. Four independent variables each significantly influenced ADAMTS-4 activity in synovial fluid (all P < 0.001): age (concordance = 0.69), presence of osteoarthritis (OA) (concordance = 0.66), level of effusion (concordance = 0.78) and inflammation (concordance = 0.68). Not only did effusion influence the amount of ADAMTS-4 activity most strongly, but it also did this in an ordered manner (P < 0.001). CONCLUSIONS: The main finding of this study is that ADAMTS-4 levels in synovial fluid are most strongly correlated with inflammation and severity of effusion in the knee. Further study is required to determine if it could provide a useful tool to aid clinical diagnoses, indicate treatment, to monitor progression of joint degeneration or OA or alternatively the success of treatment.
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Proteínas ADAM/análisis , Artropatías/enzimología , Traumatismos de la Rodilla/enzimología , Osteoartritis de la Rodilla/enzimología , Procolágeno N-Endopeptidasa/análisis , Líquido Sinovial/química , Proteína ADAMTS4 , Adulto , Biomarcadores/análisis , Transferencia Resonante de Energía de Fluorescencia , Humanos , Persona de Mediana EdadRESUMEN
OBJECTIVE: Kashin-Beck Disease (KBD) is a rare and severe osteoarthropathy endemic to China. We evaluated the frequency and patterns of hand radiographic osteoarthritis (rOA) in adults with and without KBD. METHODS: Han Chinese (N = 438) from Yongshou County of central China underwent right hand radiography for determining case status. Presence of KBD was based on characteristic radiographic deformities of articular ends of bones including articular surface depression, carpal crowding, any subchondral bone deformities in the proximal end of phalanges or first metacarpal bone, or the distal ends of metacarpal bones 2-5, and any bony enlargement with deformity of the distal ends of phalanges. Hand rOA severity was determined by osteophyte (OST), joint space narrowing (JSN), and Kellgren and Lawrence (KL) grades. RESULTS: This study included 127 KBD and 311 non-KBD adults of similar mean age (39 years) and body mass index (BMI) (21 kg/m(2)). Inter- and intra-rater reliability for radiographic determination of case status and rOA features was high (kappa 0.72-0.96). Compared to non-KBD, KBD adults had significantly more severe hand rOA of the thumb, distal interphalangeal (DIP), proximal interphalangeal (PIP) and metacarpophalangeal (MCP) joints. Only KBD adults had end-stage carpometacapal (CMC) disease. In KBD, DIPs and PIPs were more affected than MCPs and the frequency of OSTs was significantly higher in PIPs than DIPs. CONCLUSIONS: Compared with age-matched adults from the same area and farming occupation, KBD hand rOA was more widespread and severe, particularly of PIPs and CMCs. The ability to differentiate adult KBD from non-KBD hand rOA will facilitate genetic analyses of the vast majority of affected individuals.
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Articulaciones de la Mano/diagnóstico por imagen , Enfermedad de Kashin-Beck/diagnóstico por imagen , Osteoartritis/diagnóstico por imagen , Adulto , Femenino , Articulaciones de los Dedos/diagnóstico por imagen , Humanos , Enfermedad de Kashin-Beck/complicaciones , Masculino , Persona de Mediana Edad , Osteoartritis/etiología , Radiografía , Índice de Severidad de la Enfermedad , Pulgar/diagnóstico por imagenRESUMEN
The annulus fibrosus of the intervertebral disc unites adjacent vertebral bodies along the length of the spine and provides tensile resistance towards compressive, twisting and bending movements arising through gait. It consists of a nested series of oriented collagenous lamellae, arranged in cross-ply circumferentially around the nucleus pulposus. The organisation of oriented collagen in the annulus is established during foetal development by an identical arrangement of oriented fibroblasts that are precisely organised into cell sheets, or laminae. These provide a template for ordered deposition of extracellular matrix material on cell surfaces, by means of a poorly understood mechanism involving the actin cytoskeleton. In this study, we investigate the role of two cell surface heparan sulphate proteoglycans (HSPGs), glypican-6 and syndecan-4, in the matrix assembly process in the developmental rat intervertebral disc. We compare their expression patterns with those of heparan sulphate and the interactive, cell-surface adhesive glycoprotein, fibronectin, and relate these to the stage-specific collagenous architectures present within the annulus at both light and electron microscopic levels. We show that both proteoglycans are strongly associated with the development, growth and aging of the intervertebral disc. Furthermore, the immunohistochemical labelling patterns suggest that syndecan-4, in particular, plays a potentially-significant role in annulus formation. We propose that this HSPG mediates interaction between the actin cytoskeleton and nascent extracellular matrix in the lamellar organisation of annulus tissue. These data add considerably towards an understanding of how cells organise and maintain complex, oriented extracellular matrices and has particular clinical relevance to the fields of tissue engineering and repair.
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Matriz Extracelular/metabolismo , Disco Intervertebral/metabolismo , Sindecano-4/metabolismo , Animales , Diferenciación Celular/fisiología , Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , Ratas Wistar , Ingeniería de Tejidos/métodosRESUMEN
OBJECTIVES: To identify changes in the expression patterns of enzymes involved in chondroitin sulfate (CS) glycosaminoglycan (GAG) metabolism in articular cartilage proteoglycan (PG) isolated from adolescent patients with Kashin-Beck disease (KBD). METHODS: Samples of articular cartilage were divided into two groups: Control samples (from five normal children), and KBD samples (from five KBD children) aged 3-12 years old. The morphology and pathology of hand joint cartilage were examined by histochemical staining. The localization and expression patterns of enzymes involved in CS GAG metabolism (i.e., PAPS synthetase 2 (PAPSS2), PAPS transporter 1 (PAPST1), Carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferases 15 (CHST15), Arylsulfatase B (ARSB) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS)) were performed using immuno-histochemical analyses. Positive immunostaining in articular cartilage was semi-quantified. RESULTS: Reduced aggrecan staining was observed in KBD samples compared with the control samples. The percentages of positive staining for the anabolic enzymes PAPSS2, PAPST1 and CHST15 in the upper and middle zones of KBD samples were significantly lower than that found in the Controls. In contrast, the percentages of positive staining in KBD samples for the catabolic enzymes ARSB and GALNS were significantly higher than the control samples. However, the staining for all of these GAG metabolism enzymes were hardly observed in the deep zones of KBD cartilage, suggesting that significant cell death and necrosis had occurred in this region. CONCLUSIONS: Our results indicate that alterations of enzymes involved in articular cartilage CS GAG metabolism on PGs in the articular cartilage play an important role in the onset and pathogenesis of KBD in adolescent children.
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Cartílago Articular/metabolismo , Cartílago Articular/patología , Sulfatos de Condroitina/metabolismo , Enfermedad de Kashin-Beck/metabolismo , Agrecanos/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Enfermedad de Kashin-Beck/etiología , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Complejos Multienzimáticos/metabolismo , Proteoglicanos/metabolismo , Sulfato Adenililtransferasa/metabolismo , Transportadores de Sulfato , Sulfotransferasas/metabolismoRESUMEN
Chondroitin sulphate chains on cell and extracellular matrix proteoglycans play important regulatory roles in developing systems. Specific, developmentally regulated, sulphation motifs within the chondroitin glycosaminoglycan structure may help bind, sequester or present bioactive signalling molecules to cells thus modulating their behaviour. Using monoclonal antibodies 3B3(-), 4C3, 6C3 and 7D4, we have mapped the distribution of different chondroitin sulphation epitopes in a rat intervertebral disc developmental series. The sulphation epitopes had complex, dynamic and specific distributions in the disc and vertebral tissues during their differentiation, growth and ageing. At embryonic day [E]15, prior to disc differentiation, 4C3 and 7D4 occurred within the cellular disc condensations whilst 6C3 was present in the notochordal sheath. At E17, post disc differentiation, 4C3 and 7D4 occurred within the nucleus pulposus, inner annulus and vertebral bodies; 3B3(-) in the nucleus, inner annulus, annulus/vertebral body interface and perichondrium; and 6C3, ventrally, within the perichondrium. At E19, 3B3(-), 4C3 and 7D4 became further restricted to the nucleus, inner annulus, annulus/vertebral body interface and perichondrium. Prior to birth, all four epitopes occurred within the inner annulus and nucleus, with 6C3 and 7D4 also occurring within the future end-plate. Postnatal expression of the sulphation epitopes was more widespread in the disc and also within the growth plate. At 4 months, the epitopes were associated with chondrocyte clusters within the nucleus; and at 24 months, with annular lesions. Overall, our data suggests that differential sulphation of chondroitin correlates with significant events in development, growth and aging of the rat intervertebral disc.
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Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Glicosaminoglicanos , Disco Intervertebral/química , Disco Intervertebral/embriología , Animales , Anticuerpos Monoclonales , Cartílago/embriología , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Expresión Génica , Glicosaminoglicanos/química , Glicosaminoglicanos/genética , Placa de Crecimiento/química , Placa de Crecimiento/embriología , Dominios y Motivos de Interacción de Proteínas , Ratas , Transducción de Señal , Columna Vertebral/embriologíaRESUMEN
The annulus fibrosus of the intervertebral disc is a complex, radial-ply connective tissue consisting of concentric lamellae of oriented collagen. Whilst much is known of the structure of the mature annulus, less is known of how its complex collagenous architecture becomes established; an understanding of which could inform future repair/regenerative strategies. Here, using a rat disc developmental series, we describe events in the establishment of the collagenous framework of the annulus at light and electron microscopic levels and examine the involvement of class I and II small leucine rich proteoglycans (SLRPs) in the matrix assembly process. We show that a period of sustained, ordered matrix deposition follows the initial cellular differentiation/orientation phase within the foetal disc. Fibrillar matrix is deposited from recesses within the plasma membrane into compartments of interstitial space within the outer annulus - the orientation of the secreted collagen reflecting the initial cellular orientation of the laminae. Medially, we demonstrate the development of a reinforcing 'cage' of collagen fibre bundles around the foetal nucleus pulpous. This derives from the fusion of collagen bundles between presumptive end-plate and inner annulus. By birth, the distinct collagenous architectures are established and the disc undergoes considerable enlargement to maturity. We show that fibromodulin plays a prominent role in foetal development of the annulus and its attachment to vertebral bodies. With the exception of keratocan, the other SLRPs appear associated more with cartilage development within the vertebral column, but all become more prominent within the disc during its growth and differentiation.
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Colágeno/metabolismo , Tejido Conectivo/metabolismo , Disco Intervertebral/metabolismo , Disco Intervertebral/ultraestructura , Animales , Cartílago/metabolismo , Cartílago/ultraestructura , Diferenciación Celular , Condrogénesis , Tejido Conectivo/ultraestructura , Desarrollo Embrionario , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibromodulina , Disco Intervertebral/embriología , Disco Intervertebral/crecimiento & desarrollo , Microscopía Electrónica , Proteoglicanos/metabolismo , Ratas , Ratas Wistar , Columna Vertebral/crecimiento & desarrollo , Columna Vertebral/metabolismoRESUMEN
AIMS: Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) present in the corneal stroma where it is thought to regulate collagen fibril diameter. In this study we investigated the distribution of KS in normal and keratoconic corneas. METHODS: Four normal, one mild, and four severe keratoconic corneas were used for the study. Distribution of keratan sulphate proteoglycans (KS-PG) was investigated using a primary monoclonal antibody (5-D-4) that recognizes disulphated disaccharides in the poly-N-acetyllactosamine repeats of KS. The immuno-reactivity of 5-D-4 was analyzed by immunohistochemistry and immuno-electron microscopy. RESULTS: Immuno-histochemistry showed diffuse 5-D-4 staining in keratoconic cornea compared to the punctuate staining in normal corneas. In the single cornea with mild keratoconus, immunogold microscopy revealed a very high density of KS-PG staining, especially in the posterior stroma, compared to severe keratoconic and normal cornea. The amount of KS-PG in the stroma in severe keratoconus was slightly less compared to the normal cornea. In the mild keratoconic cornea, a higher quantity of KS-PG was present around the keratocytes. In severe keratoconic corneas, a higher quantity of KS-PG was present within the keratocytes compared to normal cornea. CONCLUSIONS: The finding of an altered expression of KS in our keratoconic corneas, in particular the strong expression of KS in keratocytes, is in keeping with reports of an altered expression of proteoglycan metabolism in keratoconus. KS-PG plays an important role in stromal collagen fibril assembly and a dysregulation of KS-PG synthesis or catabolism could explain changes in collagen fibril spacing and diameter, which we have reported elsewhere.
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Córnea/metabolismo , Sulfato de Queratano/metabolismo , Queratocono/metabolismo , Polisacáridos/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales , Lámina Limitante Anterior/metabolismo , Lámina Limitante Anterior/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Córnea/ultraestructura , Sustancia Propia/metabolismo , Sustancia Propia/ultraestructura , Lámina Limitante Posterior/metabolismo , Lámina Limitante Posterior/ultraestructura , Epitelio Corneal/metabolismo , Epitelio Corneal/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Sulfato de Queratano/ultraestructura , Queratocono/patología , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Polisacáridos/inmunología , Polisacáridos/ultraestructura , Sulfatos , Adulto JovenRESUMEN
OBJECTIVE: To assess the relative efficacy of three different omega-3 (n-3) polyunsaturated fatty acids (PUFAs) in suppressing the mRNA levels for important proteins involved in the etiology of osteoarthritis (OA). METHODS: A model cell culture system (bovine chondrocytes) was used. Inflammatory factors and enzymes involved in OA were induced by exposure of the chondrocyte cultures to interleukin-1alpha (IL-1alpha). The effect of pre-incubating cultures with various amounts of exogenous fatty acids on subsequent levels of mRNAs was assessed by reverse transcription-polymerase chain reactions (RT-PCR). RESULTS: Exposure of cultures to IL-1alpha induced expression of the cartilage proteinases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)-4 and ADAMTS-5, cyclooxygenase (COX)-2, the matrix metalloproteinase (MMP)-3 and the inflammatory cytokines IL-1alpha, interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). n-3 PUFAs were able to reduce the levels of mRNA for ADAMTS-4, ADAMTS-5, MMP-3, MMP-13, COX-2 (but not COX-1), IL-1alpha, IL-1beta and TNF-alpha. Eicosapentaenoic acid (EPA) was the most effective, followed by docosahexaenoic (DHA) and then alpha-linolenic (ALA) acid. The n-6 PUFA, arachidonic acid (AA) had no effect. CONCLUSION: These results show that omega-3 (n-3) PUFAs cause a reduction in the mRNA levels for various proteins known to be important in the pathology of OA. They provide a molecular explanation, at least in part, for beneficial effects of dietary omega-3 PUFAs for the amelioration of symptoms of the disease. The relative efficacy of EPA suggests that this omega-3 PUFA may be especially useful for dietary supplementation in patients with OA.
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Condrocitos/metabolismo , Ácidos Grasos Omega-3/farmacología , Osteoartritis/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas ADAM/metabolismo , Animales , Carpo Animal , Cartílago Articular/metabolismo , Bovinos , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interleucina-1alfa/farmacología , Ácido Láctico/biosíntesis , Metaloproteinasas de la Matriz/metabolismo , Osteoartritis/etiología , Osteoartritis/prevención & control , ARN Mensajero/metabolismoRESUMEN
Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid. We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer. We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts. We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein. Three link proteins were identified in normal canine articular cartilage. These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.
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Cartílago Articular/análisis , Proteínas de la Matriz Extracelular , Proteínas/análisis , Proteoglicanos , Membrana Sinovial/análisis , Animales , Cartílago Articular/metabolismo , Bovinos , Células Cultivadas , Perros , Electroforesis en Gel de Poliacrilamida , Masculino , Peso Molecular , Biosíntesis de Proteínas , Membrana Sinovial/metabolismoRESUMEN
Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b-HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan-aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.
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Cartílago Articular/ultraestructura , Membrana Celular/ultraestructura , Matriz Extracelular/ultraestructura , Ácido Hialurónico/análisis , Proteoglicanos/análisis , Animales , Cartílago Articular/citología , Bovinos , Hialuronoglucosaminidasa , Sulfato de Queratano/análisis , Streptomyces/enzimología , Grabación en VideoRESUMEN
Tendons are dense, fibrous connective tissues that are responsible for transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects function) is very common, both within the sporting arena and in the workplace. Importantly, proteoglycans are essential components of the tendon extracellular matrix (ECM) and changes in their expression and metabolism/turnover have been associated with tendinopathy. Within tendons, the small leucine-rich proteoglycans (SLPRs), decorin, fibromodulin, lumican and keratocan predominate within tensional regions, while in tendon fibrocartilage, increased concentrations of proteoglycans common to the articular cartilage phenotype are present, including aggrecan, biglycan and proteoglycan 4. However, the rate of proteoglycan turnover within tendon is markedly higher than that of cartilage, mediated via the "aggrecanases," which are constitutively active in the tendon matrix. Data suggest that this increased proteoglycan turnover is likely to be required to maintain normal tendon homeostasis, with perturbations in proteoglycan metabolism contributing to tissue dysfunction. Thus, future studies aimed at furthering our fundamental knowledge of tendon proteoglycan metabolism in health and disease are important in the development of improved treatments for tendon disorders.
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Proteoglicanos/metabolismo , Tendones/metabolismo , Matriz Extracelular/metabolismo , Fibrocartílago/metabolismo , Humanos , Estrés Mecánico , Tendinopatía/metabolismoRESUMEN
Destruction of articular cartilage is a key feature of a number of arthritides, osteoarthritis prominent among them. Aggrecan degradation, caused by increased activity of proteolytic enzymes that degrade macromolecules in the cartilage extracellular matrix, is followed by irreversible collagen degradation. The degradation of aggrecan is mediated by various matrix proteinases, mainly the aggrecanases, multidomain metalloproteinases belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family. There has been much interest in the possible role of these aggrecanases, mainly ADAMTS4 and ADAMTS5, as therapeutic targets in osteoarthritis. There is still debate which of them is the major aggrecanase in osteoarthritis, however, as well as major issues concerning how they are regulated, with possible discrepancies between murine models and results obtained using human osteoarthritis tissue. This review discusses some recent data regarding the regulation of ADAMTS4 and ADAMTS5 gene expression in osteoarthritis, with emphasis on the role of proinflammatory cytokines in driving these enzymes, and of the transcription factor NFkappaB in mediating their expression.
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Proteínas ADAM/fisiología , Osteoartritis/enzimología , Procolágeno N-Endopeptidasa/fisiología , Proteína ADAMTS4 , Proteína ADAMTS5 , Citocinas/fisiología , Sistemas de Liberación de Medicamentos , Expresión Génica , Humanos , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Membrana Sinovial/enzimologíaRESUMEN
Methods of isolating the water insoluble 'link proteins' from preparations of bovine nasal cartilage proteoglycan aggregates have been investigated. Upon chromatography on Sepharose 4B in 0.1% sodium dodecyl sulfate, the 'link proteins' are found in a discrete, included peak, whereas the bulk of the proteoglycan emerges at the void volume. Some low molecular weight proteoglycan is associated with the 'link proteins'. An alternative procedure, i.e. chromatography on DEAE-cellulose in sodium dodecyl sulfate, has also been examined. In 0.1% sodium dodecyl sulfate, proteoglycan monomer is not absorbed by the column, whereas 'link proteins' are. Subsequently, the 'link proteins' with a minor fraction of the proteoglycan are eluted with 1.0% sodium dodecyl sulfate. Both procedures serve to separate the 'link proteins' from the bulk of proteoglycan present in an aggregate preparation, but additional steps are necessary to achieve homogeneity. Thus a 'link protein' preparation, fractionated from proteoglycan aggregate by equilibrium density gradient centrifugation under dissociative conditions, can be finally purified by chromatography on DEAE-cellulose in sodium dodecyl sulfate.
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Cartílago/análisis , Proteínas/aislamiento & purificación , Aminoácidos/análisis , Animales , Bovinos , Cromatografía en Agarosa , Cromatografía DEAE-Celulosa , Hexosaminas/análisis , Nariz , Proteoglicanos , Dodecil Sulfato de SodioRESUMEN
Perineuronal nets (PNNs) are specialized extracellular matrix aggregates surrounding distinct neuronal populations and regulating synaptic functions and plasticity. Previous findings showed robust PNN decreases in amygdala, entorhinal cortex and prefrontal cortex of subjects with schizophrenia (SZ), but not bipolar disorder (BD). These studies were carried out using a chondroitin sulfate proteoglycan (CSPG) lectin marker. Here, we tested the hypothesis that the CSPG aggrecan, and 6-sulfated chondroitin sulfate (CS-6) chains highly represented in aggrecan, may contribute to these abnormalities. Antibodies against aggrecan and CS-6 (3B3 and CS56) were used in the amygdala of healthy control, SZ and BD subjects. In controls, aggrecan immunoreactivity (IR) was observed in PNNs and glial cells. Antibody 3B3, but not CS56, also labeled PNNs in the amygdala. In addition, dense clusters of CS56 and 3B3 IR encompassed CS56- and 3B3-IR glia, respectively. In SZ, numbers of aggrecan- and 3B3-IR PNNs were decreased, together with marked reductions of aggrecan-IR glial cells and CS-6 (3B3 and CS56)-IR 'clusters'. In BD, numbers of 3B3-IR PNNs and CS56-IR clusters were reduced. Our findings show disruption of multiple PNN populations in the amygdala of SZ and, more modestly, BD. Decreases of aggrecan-IR glia and CS-6-IR glial 'clusters', in sharp contrast to increases of CSPG/lectin-positive glia previously observed, indicate that CSPG abnormalities may affect distinct glial cell populations and suggest a potential mechanism for PNN decreases. Together, these abnormalities may contribute to a destabilization of synaptic connectivity and regulation of neuronal functions in the amygdala of subjects with major psychoses.
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Agrecanos/metabolismo , Amígdala del Cerebelo/metabolismo , Trastorno Bipolar/metabolismo , Sulfatos de Condroitina/metabolismo , Neuroglía/metabolismo , Esquizofrenia/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana EdadRESUMEN
In this paper we report the use of immunological methods for specifically detecting and determining proteoglycan in cartilage and other connective tissues. Antibodies (polyclonal and monoclonal) have been raised against specific components of cartilage proteoglycan aggregates (i.e., proteoglycan monomer and link protein). Radioimmunoassay procedures and immunohistochemical procedures have been developed and used to demonstrate the occurrence of cartilage-like proteoglycan and link protein in bovine aorta. Similarly, immunofluorescent studies have been used to analyze proteoglycan distribution in skin. Using antibodies specific for chondroitin-4-sulfated proteoglycan, their presence was demonstrated in dermal connective tissue and connective tissue surrounding nerve and muscle sheaths. However, chondroitin-4-sulfated proteoglycan was completely absent in the epidermis of skin and areas surrounding invaginating hair follicles. These immunological procedures are currently being used to complement conventional biochemical analyses of proteoglycans found in different connective tissue matrices.
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Tejido Conectivo/análisis , Proteínas de la Matriz Extracelular , Proteoglicanos/análisis , Animales , Anticuerpos Monoclonales , Cartílago/análisis , Bovinos , Sulfatos de Condroitina/análisis , Técnica del Anticuerpo Fluorescente , Proteínas/análisis , Radioinmunoensayo/métodos , RatasRESUMEN
Monoclonal antibodies that recognize carbohydrate epitopes found in keratan sulfate glycosaminoglycan chains identified both intracytoplasmic and cell-surface carbohydrates of human keratinocytes. These carbohydrates were detected, using indirect immunoperoxidase methods, both in sections of paraffin-embedded tissues and in intact cultured keratinocytes. Of the seven anti-keratan sulfate monoclonal antibodies used in this study, five detected significant amounts of epitopes associated with keratinocytes. This indicates that only certain, specific types of keratan sulfate-like carbohydrates were expressed by these cells. The extent and localization of keratan sulfate-like carbohydrates appeared to be closely related to the differentiation status of cultured keratinocytes. These epitopes were very weakly expressed on surfaces of all monolayer keratinocytes, but flattened, suprabasal cells in high Ca++ cultures strongly expressed keratan sulfate-like carbohydrates on their surfaces. A much larger population of cultured keratinocytes expressed intracellular keratan sulfate-like carbohydrates identified by the same five antibodies that detected surface epitopes. In monolayer cells, keratan sulfate-like carbohydrates were predominantly found in a broad perinuclear zone. In addition, three of the five immunoreactive antibodies detected epitopes that appeared at cell boundaries, specifically at sites of close cell-to-cell contact. Thus, molecules bearing carbohydrates recognized by anti-keratan sulfate antibodies appear at developmentally important stages of keratinocyte differentiation, indicating that these carbohydrates may serve as markers for molecules important in the differentiation of human keratinocytes.
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Carbohidratos/inmunología , Queratinocitos/inmunología , Anticuerpos Monoclonales/análisis , Especificidad de Anticuerpos , Diferenciación Celular , Epítopos , Humanos , Inmunohistoquímica , Sulfato de Queratano/inmunología , Queratinocitos/citologíaRESUMEN
Oligonucleotide primers which were designed based on identical peptide sequences flanking the interglobular domain (IGD) of human, bovine and rat aggrecan were used in RT-PCR reactions containing human, porcine, equine, bovine and ovine cartilage RNA. Novel cDNAs encoding the IGD of the latter four species were obtained and sequenced. The deduced amino acid sequences for these cDNAs were aligned and compared with those described for six other species. Amino acid sequences surrounding the major proteolytic cleavage sites in the IGD are highly conserved, with some species-specific substitutions. Similarly, known sites of keratan sulfate attachment in the IGD are highly conserved in all species. The results provide essential amino acid sequence data for species commonly used in model systems of cartilage degeneration.
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Cartílago Articular/química , Proteínas de la Matriz Extracelular , Sulfato de Queratano/metabolismo , Estructura Terciaria de Proteína , Proteoglicanos/genética , Homología de Secuencia de Aminoácido , Agrecanos , Animales , Bovinos , Clonación Molecular , Secuencia Conservada , Perros , Caballos , Humanos , Lectinas Tipo C , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteoglicanos/química , Conejos , Ratas , Alineación de Secuencia , Ovinos , PorcinosRESUMEN
The increased catabolism of the cartilage proteoglycan aggrecan is a principal pathological process which leads to the degeneration of articular cartilage in arthritic joint diseases. The consequent loss of sulphated glycosaminoglycans, which are intrinsic components of the aggrecan molecule, compromises both the functional and structural integrity of the cartilage matrix and ultimately renders the tissue incapable of resisting the compressive loads applied during joint articulation. Over time, this process leads to irreversible cartilage erosion. In situ degradation of aggrecan is a proteolytic process involving cleavage at specific peptide bonds located within the core protein. The most well characterised enzymatic activities contributing to this process are engendered by zinc-dependent metalloproteinases. In vitro aggrecanolysis by matrix metalloproteinases (MMPs) has been widely studied; however, it is now well recognised that the principal proteinases responsible for aggrecan degradation in situ in articular cartilage are the aggrecanases, two recently identified isoforms of which are members of the 'A Disintegrin And Metalloproteinase with Thrombospondin motifs' (ADAMTS) gene family. In this review we have described: (i) the development of monoclonal antibody technologies to identify catabolic neoepitopes on aggrecan degradation products; (ii) the use of such neoepitope antibodies in studies designed to characterise and identify the enzymes responsible for cartilage aggrecan metabolism; (iii) the biochemical properties of soluble cartilage aggrecanase(s) and their differential expression in situ; and (iv) model culture systems for studying cartilage aggrecan catabolism. These studies have clearly established that 'aggrecanase(s)' is primarily responsible for the catabolism and loss of aggrecan from articular cartilage in the early stages of arthritic joint diseases that precede overt collagen catabolism and disruption of the tissue integrity. At later stages, when collagen catabolism is occurring, there is evidence for MMP-mediated degradation of the small proportion of aggrecan remaining in the tissue, but this occurs independently of continued aggrecanase activity. Furthermore, the catabolism of link proteins by MMPs is also initiated when overt collagen degradation is evident.
Asunto(s)
Cartílago Articular/metabolismo , Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Proteoglicanos/metabolismo , Animales , Endopeptidasas/genética , Humanos , Modelos Biológicos , Proteoglicanos/antagonistas & inhibidoresRESUMEN
The chondrocytes of articular cartilage synthesize a number of proteinases which are capable of degrading the component molecules of this specialized extracellular matrix. The use of class-specific proteinase inhibitors indicates that major activities responsible for catabolism of proteoglycan (aggrecan) and collagen are attributable to zinc-dependent metalloproteinases. In this study, we have compared the mRNA expression profiles of two matrix metalloproteinases (MMP-3 and MMP-13) and five disintegrin-metalloproteinases (ADAM-10, ADAM-9, ADAM-15, TNF-alpha-converting enzyme and decysin) by chondrocytes (human, porcine and bovine) from fresh cartilage and in cartilage explant cultures and isolated cells cultured in monolayer or in agarose gels. Such cultures were maintained in the presence or absence of interleukin-1 (IL-1) or all-trans-retinoic acid, two agents which promote cartilage matrix degradation in vitro. Whereas transcripts for all metalloproteinases examined were detected in chondrocytes from human osteoarthritic cartilage in monolayer cultures, mRNAs for ADAM-15 and decysin were not present in fresh osteoarthritic human cartilage or explant cultures. Similarly, expression of porcine and bovine metalloproteinase mRNAs varied with different culture conditions. Novel cDNA sequences obtained for porcine and bovine MMP-3 and MMP-13, porcine ADAM-10, porcine and bovine ADAM-9 and porcine TACE confirmed expression of mRNAs for these molecules by articular chondrocytes. Quantitative RT-PCR analysis was used to determine the effects of IL-1 and retinoic acid on metalloproteinase mRNA levels in human chondrocytes cultured in monolayer and in porcine chondrocytes cultured in agarose. For the MMPs, IL-1 treatment resulted in an approximately two to threefold increase in human and porcine MMP-3 and MMP-13 mRNAs, while retinoic acid treatment caused a statistically significant increase in human MMP-3 mRNA levels, but no significant change in transcript levels for porcine MMP-3 nor human or porcine MMP-13. The mRNA levels for ADAM-15 were elevated in human monolayer chondrocytes exposed to IL-1 or retinoic acid, while transcripts levels for TNF-alpha converting enzyme were increased in response to retinoic acid. In contrast, ADAM-9 mRNA levels were decreased in human monolayer chondrocytes exposed to IL-1 or retinoic acid. The results demonstrate that chondrocyte metalloproteinase expression can vary dependent on cell environment in situ and in vitro, and information on chondrocyte MMP and ADAM gene expression following cytokine (IL-1) or retinoid stimulation.
Asunto(s)
Cartílago Articular/enzimología , Condrocitos/enzimología , Desintegrinas , Interleucina-1/farmacología , Metaloendopeptidasas/genética , Tretinoina/farmacología , Proteínas ADAM , Proteína ADAM10 , Proteína ADAM17 , Anciano , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Cartílago Articular/citología , Bovinos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Colagenasas/genética , Medios de Cultivo , ADN Complementario , Expresión Génica , Humanos , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasas de la Matriz/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , ARN Mensajero , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Elevated concentrations of interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) in the synovial fluids and serum of patients with arthritis have been implicated in the joint tissue destruction associated with these conditions, however studies conducted to date on the role and effects of IL-6 in the process of cartilage proteoglycan (aggrecan) catabolism are disparate. In the present study, bovine articular cartilage explants were maintained in a model organ culture system in the presence or absence of IL-1alpha or TNF-alpha, and under co-stimulation with or without IL-6 and/or sIL-6R. After measuring proteoglycan loss from the explants, the proteolytic activity and expression profiles of aggrecanase(s) was assessed for each culture condition. Stimulation of cartilage explants with IL-6 and/or sIL-6R potentiated aggrecan catabolism and release above that seen in the presence of IL-1alpha or TNF-alpha alone. This catabolism was associated with aggrecanase (but not MMP) activity, with correlative mRNA expression for aggrecanase-2.